Anemoside B4 targets NEK7 to inhibit NLRP3 inflammasome activation and alleviate MSU-induced acute gouty arthritis by modulating the NF-κB signaling pathway

Abstract

Background: Acute gouty arthritis is a metabolic disorder caused by monosodium urate (MSU) accumulation, leading to NLRP3 inflammasome activation and joint inflammation. Anemoside B4 (B4), a pentacyclic triterpenoid saponin, exerts significant anti-inflammatory effects. However, the precise molecular mechanisms underlying its therapeutic action, particularly its targeting of key components in NLRP3 inflammasome activation, remain unclear.

Purpose: The aim of this study was to elucidate the therapeutic mechanisms and target of B4 in treating MSU-induced macrophage pyroptosis and acute gouty arthritis, focusing specifically, on its interaction with NEK7, a critical regulator of NLRP3 inflammasome activation.

Methods: Comprehensive in vitro and in vivo methods were employed to examine the effects and mechanisms of B4. In vitro analyses included Western blot, co-immunoprecipitation (Co-IP), and immunofluorescence assays to assess NLRP3 inflammasome components and NEK7-NLRP3 interactions. The binding of B4 to NEK7 was evaluated using molecular docking, surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), NEK7 gene silencing, and site-specific amino acid mutation experiments. In vivo, MSU-induced acute gouty arthritis mouse models and NEK7 knockdown mouse models were used to demonstrate the therapeutic effects and specificity of B4.

Results: This study provides the first evidence that B4 significantly inhibits MSU-induced inflammation and pyroptosis in macrophages by directly targeting NEK7 and disrupting the NEK7-NLRP3 complex, thereby reducing NLRP3 inflammasome activation. Additionally, B4 effectively suppressed MSU-induced ROS production, mitochondrial damage, and NF-κB activation. In vivo, B4 alleviated symptoms of acute gouty arthritis, reduced NLRP3 expression, and demonstrated specificity for NEK7 in NEK7 knockdown mouse models.

Conclusion: This study highlights B4 as an effective inhibitor of NLRP3 inflammasome activation by directly targeting NEK7, thereby mitigating inflammation and pyroptosis in acute gouty arthritis. These findings position B4 as a prospective therapeutic candidate for the management of acute gouty arthritis, providing insights into its molecular targets and mechanisms.

Keywords: Acute gouty arthritis; Anemoside B4; NEK7; NF-κB; NLRP3 inflammasome; Pyroptosis.